Abstract A128: Identification of peptide inhibitors of KRAS G12D and Q61H mutant protein for cancer therapy

Abstract The G12D mutation in the KRAS gene is one of the most common oncogenic mutations and acts as an important oncogenic mechanism in various cancers. In particular, this mutation, which is found in about 40% of pancreatic cancers, about 13% of colon cancers, and about 7% of lung adenocarcinomas, significantly reduces GTPase activity and induces persistent KRAS signaling. KRAS G12D persistently activates downstream signaling pathways, including the RAF-MEK-ERK and PI3K-AKT pathways, to promote cell proliferation, survival, invasion, and metastasis. We conducted experiments to find peptides that selectively bind to KRAS protein containing G12D/Q61H mutations using screening of phage library. Through screening analysis of the identified sequences, phage clones showing high binding values in phage binding analysis were selected and the peptide (named KRASpep-2) was synthesized in cyclic form. KRASpep-2 bound to the recombinant KRAS G12D/Q61H protein at higher levels than to wild-type KRAS protein. In addition, KRASpep-2 pulled-down KRAS G12D in the cell lysates of AsPC-1 tumor cells bearing KRAS G12D mutation, while little pull-dwon being observed using the cell lysate of H460 tumor cell bearing KRAS Q61H, suggesting that G12D mutation is the key binding determinant For efficient introduction into cells, KRASpep-2 was linked to RRRRRRR peptide (named R7), a cell-penetrating peptide, via, GGG linker. While KRASpep-2-GGG-R7 did not exert cytotoxicity in AsPC-1 cells, KRASpep-2-GGG-R7 linked with RLAA motif, a E3 ligase ligand, at its amino terminal exerted cytotoxicity in AsPC-1 cells but not, in HEK293 normal cells RLAA-KRASpep-2-GGG-R7 decreased the levels of KRAS G12D in AsPC-1 cells as a ProTac probe and those of phospho-AKT. It remains to be studied whether RLAA-KRASpep-2-GGG-R7 degrades KRAS G12D mutant protein of tumor cells in the tumor microenvironment, presenting it as potential cancer therapeutics targeting tumors with KRAS mutation. Citation Format: Dong Gyun Jo, Byungheon Lee. Identification of peptide inhibitors of KRAS G12D and Q61H mutant protein for cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A128.